Compositions and methods for enhancing coagulation factor VIII function

ABSTRACT

Factor VIII variants and methods of use thereof are disclosed.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/468,108, filed Aug. 25, 2014, which is a continuation applicationof U.S. application Ser. No. 13/437,486, filed Apr. 2, 2012, now U.S.Pat. No. 8,816,054, which is a continuation of International ApplicationNo. PCT/US2010/051285, filed Oct. 4, 2010, which claims the benefit ofpriority to U.S. Provisional Application No. 61/248,179, filed Oct. 2,2009, all of which applications are expressly incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference it its entirety. Said ASCII copy, created on Jun. 5, 2017, isnamed CHOP0452488_ST25.txt and is 724 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and hematology.More specifically, the invention provides novel coagulation Factor VIIIagents and methods of using the same to modulate the coagulation cascadein patients in need thereof.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

Hemophilia A (HA) is an X-linked bleeding disease resulting from afunctional FVIII deficiency, affecting 1:5000 males worldwide. Forseveral decades the HA dog model has been the most extensively used forpreclinical studies (1). Notably, in two strains of dogs the underlyingmutation consists of an inversion in intron 22 of the FVIII gene that isanalogous to the most common human mutation (2). This model faithfullyreplicates the human disease at both genotypic and phenotypic levels(3,4). To date there is no characterization of the cFVIII protein due todifficulties in purifying large amounts from canine plasma and to therelative poor performance in recombinant FVIII expression systems ingeneral. Although the cFVIII cDNA sequence has a high sequence identityto human FVIII (hFVIII) (5), adult HA dogs develop immune responses uponexposure to hFVIII that preclude the assessment of the efficacy andsafety of potential novel therapies for HA. Notably, among humans evensmall nucleotide changes in the hFVIII gene may predispose to inhibitorformation (6).

Identifying hFVIII variants that exhibit superior coagulation propertiesare highly desirable. It is an object of the invention to provide suchproteins for use as therapeutics.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, a Factor VIII (FVIII)variant which exhibits improved capacity to modulate hemostasis isprovided. In a preferred embodiment, the variant FVIII is a humanvariant, lacking most of the B domain (BDD=B domain deleted), comprisinga R1645H amino acid substitution which exhibits increased specificactivity and stability relative to human FVIII-BDD lacking saidsubstitution. In yet another embodiment, variants comprising furtherdeletions and modifications to the PACE/FURIN cleavage site are alsowithin the scope of the invention. Also provided are nucleic acidsencoding the variants described herein. Such nucleic acids areoptionally cloned into an expression vector. Host cells comprising suchexpression vectors are also encompassed by the present invention.

In yet another aspect, a pharmaceutical composition comprising theFactor VIII variant described above in a biologically compatible carrieris provided.

The invention also provides a method for treatment of a hemostasisrelated disorder in a patient in need thereof, comprising administrationof a therapeutically effective amount of the pro-coagulant variant FVIIIdescribed herein in a biologically acceptable carrier. Such disordersinclude, without limitation hemophilia A, hemophilia A with inhibitor,von Willebrand diseases, non-hemophilia subjects with inhibitors toFVIII, disorders of platelets, ADAMTS13-related diseases, bleedingassociated with trauma, injury coagulopathy, and disseminatedintravascular coagulation (DIC).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Western blot analysis of cFVIII-BDD monoclonal antibodies.Monoclonal antibodies were screened by western blot using cFVIII-BDD andthrombin activated cFVIII-BDD to determine their specificity for theheavy chain (HC) or the light chain (LC). One microgram of cFVIII oractivated cFVIII was loaded onto a reducing 4-12% NuPage gel withSeablue Plus 2 marker (Invitrogen, Carlsbad, Calif.), run at 200V for 50minutes and transferred to a nitrocellulose membrane. cFVIII wasdetected using the mouse monoclonal antibody (4 μg/ml) followed bydetection with IRDye 800CW rabbit anti-mouse IgG (H&L) (RocklandImmunochemicals, Inc., Gilbertsville, Pa.) and scanned on an OdysseyInfrared Imaging System (LI-COR Biosciences, Lincoln, Nebr.).

FIG. 2: Biochemical characterization of FVIII-BDD. (FIG. 2A) Canine (c)FVIII-BDD is predominantly synthesized as a 160 kD single chain proteinwith a smaller proportion being processed as a heterodimer. Thrombin(IIa) cleaves cFVIIIBDD at the indicated sites to yield activatedcFVIII. (FIG. 2B) Protein purity was assessed by loading 4 μg of humanFVIII-BDD (H) and cFVIIIBDD (C) on a reducing SDS-PAGE followed bystaining with Comassie blue (left; −IIa). FVIII-BDD (H or C; 800 nM)were incubated with IIa (+IIa; 5 nM) for 10 min and the resultingactivated FVIII was run on a reducing SDS-PAGE (right, +IIa). Thevarious domains of FVIII are indicated: SC: single chain, HC: heavychain, LC: light chain, A3-C1-C2 (73 kDa), A1 (50 kDa), and A2 (43 kDa)(FIG. 2C) The specific activity of cFVIII-BDD and hFVIII-BDD werecompared using a one- or two-stage aPTT in human deficient plasma. Forthe two stage assay (+IIa), FVIIIBDD (human or canine; 20 nM) in 20 mMHepes/150 mM NaCl/5 mM CaCl₂/0.01% Tween 80, pH 7.4 (assay buffer) wereintentionally activated with IIa (40 nM) for 30 sec at 25° C. ActivatedFVIII was immediately diluted into assay buffer with 0.1% albumin andthen subsequently added to the aPTT clotting assay. In either the one-(−IIa) or two-stage aPTT (+IIa) the specific activity of cFVIII-BDD was3-fold higher than hFVIII-BDD. The activation quotient was 22 for cFVIIIand 28 for hFVIII. (FIG. 2D) A purified Xase assay was used to assess A2domain stability. The Xase assay was performed by activating 20 nMcFVIII-BDD or hFVIII-BDD with 40 nM IIa for 30 seconds at 25° C. Thereaction was stopped by adding 60 nM hirudin. At various time pointsafter activation, FVIIIa (0.2 nM, final) was added to the Xase complex[hFIXa (2 nM), hFX (300 nM) and phospholipids (20 μM,phosphatidylcholine/phosphatidylserine; 75:25] and activation wasmeasured by monitoring FXa generation using a chromogenic substrate.

FIG. 3. Canine FVIII-BDD is functional and does not induce an immuneresponse in HA Dogs. FIG. 3A) Whole blood clotting time (WBCT) followingthree injections of cFVIII-BDD in an HA dog (mean±SD). The WBCTshortened within 5 minutes of the protein infusion from >45 minutes(baseline) to 13-16.5 min (normal range: 8-12 min). FIG. 3B) FVIIIantigen (blue lines) and clotting activity (red line) following IVinjection of cFVIII-BDD. For one protein infusion of the same dog,cFVIII activity was determined by Coatest assay and antigen levels weredetermined by ELISA specific for the cFVIII heavy (HC) or light chain(LC). The Coatest was performed using purified cFVIII as a standard. Oneunit is defined as 100 ng/ml. FIG. 3C) Monitoring antibody and inhibitorformation to cFVIII-BDD in HA dogs. In addition to the adult dogs,neonatal naïve animals that had not previously been exposed to normalcanine plasma were treated with cFVIII-BDD. IgG represents both IgG1 andIgG2 data.

FIG. 4. Recovery of FVIII after exposure to anti-hFVIII antibodies.Different concentrations of cFVIII-BDD or hFVIII-BDD were incubated withhuman plasma containing hFVIII-specific neutralizing antibodies (11B.U.) (George King Biomedical, Inc., Overland Park, Kans.) and residualFVIII activity was measured either immediately or after incubation at37° C. for 2 hours. One unit is defined as 100 ng/mL.

FIG. 5. Human FVIII (WT) or variant FVIII1645H and Canine FVIII (WT) orvariant cFVIII H1637R. SDS-PAGE followed by staining with Coomassie blue(left; +thrombin activation−IIa). FVIII-BDD (human or canine; 800 nM)were incubated with IIa (+IIa; 5 nM) for 10 min and the resultingactivated FVIII was run on a reducing SDS-PAGE (right, +IIa). Thevarious domains of FVIII are indicated: SC: single chain, HC: heavychain, LC: light chain, A3-C1-C2 (73 kDa), A1 (50 kDa), and A2 (43 kDa).

FIG. 6. A2-domain stability of human FVIII (WT) and variant FVIII-RH.The Xase assay was performed by activating 20 nM of hFVIII-BDD formswith 40 nM IIa for 30 seconds at 25° C. The reaction was stopped byadding 60 nM hirudin. At various time points after activation, FVIIIa(0.2 nM, final) was added to the Xase complex [hFIXa (2 nM), hFX (300nM) and phospholipids (20 μM, phosphatidylcholine/phosphatidylserine;75:25] and activation was measured by monitoring FXa generation using achromogenic substrate.

FIG. 7. Studies of PhF8^(RH) Lentivirus. (A) phF8 antigenic level inplatelets from F8^(null) mice reconstituted with marrow from linehF8.38/F8^(null) mice (#38) or from F8^(null) mice transfected witheither PhF8 or PhF8^(RH) lentivirus driven by the PF4 promoter (FIG.2C). (B) FeCl₃ carotid artery injury studies of F8^(null) micereconstituted with marrow transduced with an empty lentivirus FPW orPF4-driven hBF8, cBF8 or hBF8^(RH). Each set of 3 bars go from left(light) to right (dark): 20%, 15%, and 10% FeCl₃, 3 min injuries. N=3-5mice per arm. Mean±2 SE shown. *=p<0.01 compared to FPW. (C) Cremasterarteriole laser injury studies of WT, F8^(null), PhF8/F8^(null) andPhF8^(RH)/F8^(null) mice. Fibrin (red) and Plt (green) accumulation overthe 3 min is shown. Number of injuries and mice studied are indicated.

FIG. 8. In vivo efficacy of plasma hFVIII-HR in severe hemophilia A miceusing a mouse model of liver-specific expression by AAV vectors.Constructs utilized for recombinant FVIII-RH (top) and FVIII-WT(bottom).

FIG. 9A: Circulating human FVIII-RH and FVIII-WT antigen levels in mice12 weeks after gene transfer. Control groups: un-treated HA mice andwild-type with no human FVIII expression. FIG. 9B: Clotting activity ofhuman FVIII-RH and FVIII-WT in mice 12 weeks after gene transfer.Control groups: un-treated HA mice (PBS); Wild-type hemostaticallynormal mice with no human FVIII expression (WT). Experimental groups: 1)Hemophilia A CD4+ deficient; 2) Hemostatic normal mice (PBS or WT) N=7-8mice/group; AAV-8: 2×10¹³ vg/kg.

FIG. 10: Normalization of bleeding time in human FVIII-RH mice comparedto human FVIII-WT in mice 12 weeks after gene transfer. Control groups:PBS-treaded HA mice and hemostatic normal WT-mice.

FIG. 11: A table showing results from injury studies at the carotidartery induced by FeCl3. 3 minute observation; 2 minute injury (15%FeCl3) time=0, observe for 30 minutes.

FIG. 12: Data obtained using a laser induced thrombus model. FIG. 12A isreproduced from Ivanciu et al., Nat. Biotechnol (2011) and shows a laserinduced thrombus formation model. FIG. 12B shows the results obtainedfrom lentiviral mediated platelet restricted expression of human FVIIIRHon in vivo thrombus formation occurring via laser-induced injury. FIG.12C shows data obtained at the arteriole and the venule.

DETAILED DESCRIPTION OF THE INVENTION

Production of recombinant B-domain deleted canine factor VIII(cFVIII-BDD) unexpectedly revealed superior protein yields with 3-foldincreased specific activity and stability relative to human FVIII-BDD(hFVIII-BDD). The cFVIII-BDD is efficient at inducing hemostasis inhuman plasma containing FVIII inhibitors. Infusion of cFVIII-BDD inhemophilia A dogs resulted in correction of the disease phenotype with apharmacokinetic profile similar to clinical experience with hFVIII-BDD.Notably, immune tolerance challenges with cFVIII-BDD in young and adulthemophilia A dogs did not induce the formation of neutralizing ornon-neutralizing antibodies to cFVIII. These data indicate that theFVIII variant described herein should exhibit greater efficacy andsafety in preclinical studies of new therapies for hemophilia A.

I. Definitions

Various terms relating to the biological molecules of the presentinvention are used hereinabove and also throughout the specification andclaims.

The phrase “variant Factor VIII (FVIII)” refers to a modified FVIIIwhich has been genetically altered such that the encoded proteinexhibits a 3-fold increase in specific activity and enhanced stabilitywhen compared to unmodified FVIII. The nucleotide sequences describedherein are readily obtainable from GenBank. For human FVIII, seeAccession No. NG-011403.1. For canine FVIII, see Accession No.NM-001003212-1. cFVIII-BDD refers to a FVIII variant which lacks the Bdomain.

The phrase “hemostasis related disorder” refers to bleeding disorderssuch as hemophilia A, hemophilia A patients with inhibitory antibodies,deficiencies in coagulation Factors, VII, VIII, IX and X, XI, V, XII,II, von Willebrand factor, combined FV/FVIII deficiency, vitamin Kepoxide reductase C1 deficiency, gamma-carboxylase deficiency; bleedingassociated with trauma, injury, thrombosis, thrombocytopenia, stroke,coagulopathy, disseminated intravascular coagulation (DIC);over-anticoagulation associated with heparin, low molecular weightheparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e.FXa inhibitors); and platelet disorders such as, Bernard Souliersyndrome, Glanzman thromblastemia, and storage pool deficiency.

With reference to nucleic acids of the invention, the term “isolatednucleic acid” is sometimes used. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it originates. For example,the “isolated nucleic acid” may comprise a DNA or cDNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into theDNA of a prokaryote or eukaryote.

With respect to RNA molecules of the invention, the term “isolatednucleic acid” primarily refers to an RNA molecule encoded by an isolatedDNA molecule as defined above. Alternatively, the term may refer to anRNA molecule that has been sufficiently separated from RNA moleculeswith which it would be associated in its natural state (i.e., in cellsor tissues), such that it exists in a “substantially pure” form (theterm “substantially pure” is defined below).

With respect to protein, the term “isolated protein” or “isolated andpurified protein” is sometimes used herein. This term refers primarilyto a protein produced by expression of an isolated nucleic acid moleculeof the invention. Alternatively, this term may refer to a protein whichhas been sufficiently separated from other proteins with which it wouldnaturally be associated, so as to exist in “substantially pure” form.

The term “promoter region” refers to the transcriptional regulatoryregions of a gene, which may be found at the 5′ or 3′ side of the codingregion, or within the coding region, or within introns.

The term “vector” refers to a small carrier DNA molecule into which aDNA sequence can be inserted for introduction into a host cell where itwill be replicated. An “expression vector” is a specialized vector thatcontains a gene or nucleic acid sequence with the necessary regulatoryregions needed for expression in a host cell.

The term “operably linked” means that the regulatory sequences necessaryfor expression of a coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toeffect expression of the coding sequence. This same definition issometimes applied to the arrangement of coding sequences andtranscription control elements (e.g. promoters, enhancers, andtermination elements) in an expression vector. This definition is alsosometimes applied to the arrangement of nucleic acid sequences of afirst and a second nucleic acid molecule wherein a hybrid nucleic acidmolecule is generated.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight the compound of interest (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-99% by weight,of the compound of interest. Purity is measured by methods appropriatefor the compound of interest (e.g. chromatographic methods, agarose orpolyacrylamide gel electrophoresis, HPLC analysis, and the like).

The phrase “consisting essentially of” when referring to a particularnucleotide sequence or amino acid sequence means a sequence having theproperties of a given SEQ ID NO. For example, when used in reference toan amino acid sequence, the phrase includes the sequence per se andmolecular modifications that would not affect the basic and novelcharacteristics of the sequence.

The term “oligonucleotide,” as used herein refers to primers and probesof the present invention, and is defined as a nucleic acid moleculecomprised of two or more ribo- or deoxyribonucleotides, preferably morethan three. The exact size of the oligonucleotide will depend on variousfactors and on the particular application for which the oligonucleotideis used. The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and method of use. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides.

The probes herein are selected to be “substantially” complementary todifferent strands of a particular target nucleic acid sequence. Thismeans that the probes must be sufficiently complementary so as to beable to “specifically hybridize” or anneal with their respective targetstrands under a set of pre-determined conditions. Therefore, the probesequence need not reflect the exact complementary sequence of thetarget. For example, a non-complementary nucleotide fragment may beattached to the 5′ or 3′ end of the probe, with the remainder of theprobe sequence being complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecifically.

The term “specifically hybridize” refers to the association between twosingle-stranded nucleic acid molecules of sufficiently complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule of the invention, to thesubstantial exclusion of hybridization of the oligonucleotide withsingle-stranded nucleic acids of non-complementary sequence.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to act functionally as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product.

The primer may vary in length depending on the particular conditions andrequirements of the application. For example, in diagnosticapplications, the oligonucleotide primer is typically 15-25 or morenucleotides in length. The primer must be of sufficient complementarityto the desired template to prime the synthesis of the desired extensionproduct, that is, to be able to anneal with the desired template strandin a manner sufficient to provide the 3′ hydroxyl moiety of the primerin appropriate juxtaposition for use in the initiation of synthesis by apolymerase or similar enzyme. It is not required that the primersequence represent an exact complement of the desired template. Forexample, a non-complementary nucleotide sequence may be attached to the5′ end of an otherwise complementary primer. Alternatively,non-complementary bases may be interspersed within the oligonucleotideprimer sequence, provided that the primer sequence has sufficientcomplementarity with the sequence of the desired template strand tofunctionally provide a template-primer complex for the synthesis of theextension product.

The term “percent identical” is used herein with reference tocomparisons among nucleic acid or amino acid sequences. Nucleic acid andamino acid sequences are often compared using computer programs thatalign sequences of nucleic or amino acids thus defining the differencesbetween the two. For purposes of this invention comparisons of nucleicacid sequences are performed using the GCG Wisconsin Package version9.1, available from the Genetics Computer Group in Madison, Wis. Forconvenience, the default parameters (gap creation penalty=12, gapextension penalty=4) specified by that program are intended for useherein to compare sequence identity. Alternately, the Blastn 2.0 programprovided by the National Center for Biotechnology Information (found onthe world wide web at ncbi.nlm.nih.gov/blast/; Altschul et al., 1990, JMol Biol 215:403-410) using a gapped alignment with default parameters,may be used to determine the level of identity and similarity betweennucleic acid sequences and amino acid sequences.

II. Preparation of Variant FVIII Encoding Nucleic Acid Molecules andPolypeptides

A. Nucleic Acid Molecules

Nucleic acid molecules encoding the variant FVIII molecules of theinvention may be prepared by using recombinant DNA technology methods.The availability of nucleotide sequence information enables preparationof isolated nucleic acid molecules of the invention by a variety ofmeans. For example, nucleic acid sequences encoding a variant FVIIIpolypeptide may be isolated from appropriate biological sources usingstandard protocols well known in the art.

Nucleic acids of the present invention may be maintained as DNA in anyconvenient cloning vector. In a preferred embodiment, clones aremaintained in a plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell. Alternatively, the nucleic acids may be maintained invector suitable for expression in mammalian cells. In cases wherepost-translational modification affects coagulation function, it ispreferable to express the molecule in mammalian cells.

Variant FVIII-encoding nucleic acid molecules of the invention includecDNA, genomic DNA, RNA, and fragments thereof which may be single- ordouble-stranded. Thus, this invention provides oligonucleotides (senseor antisense strands of DNA or RNA) having sequences capable ofhybridizing with at least one sequence of a nucleic acid molecule of thepresent invention. Such oligonucleotides are useful as probes fordetecting FVIII expression.

B. Proteins

A B-domain deleted FVIII polypeptide of the present invention may beprepared in a variety of ways, according to known methods. The proteinmay be purified from appropriate sources, e.g., transformed bacterial oranimal cultured cells or tissues which express engineered FVIII byimmunoaffinity purification.

The availability of nucleic acid molecules encoding a variant FVIIIpolypeptide enables production of FVIII using in vitro expressionmethods known in the art. For example, a cDNA or gene may be cloned intoan appropriate in vitro transcription vector, such as pSP64 or pSP65 forin vitro transcription, followed by cell-free translation in a suitablecell-free translation system, such as wheat germ or rabbit reticulocytelysates. In vitro transcription and translation systems are commerciallyavailable, e.g., from Promega Biotech, Madison, Wis. or BRL, Rockville,Md.

Alternatively, according to a preferred embodiment, larger quantities ofFVIII may be produced by expression in a suitable prokaryotic oreukaryotic expression system. For example, part or all of a DNA moleculeencoding variant Factor VIII for example, may be inserted into a plasmidvector adapted for expression in a bacterial cell, such as E. coli or amammalian cell such as CHO or Hela cells. Alternatively, in a preferredembodiment, tagged fusion proteins comprising FVIII can be generated.Such FVIII-tagged fusion proteins are encoded by part or all of a DNAmolecule, ligated in the correct codon reading frame to a nucleotidesequence encoding a portion or all of a desired polypeptide tag which isinserted into a plasmid vector adapted for expression in a bacterialcell, such as E. coli or a eukaryotic cell, such as, but not limited to,yeast and mammalian cells. Vectors such as those described abovecomprise the regulatory elements necessary for expression of the DNA inthe host cell positioned in such a manner as to permit expression of theDNA in the host cell. Such regulatory elements required for expressioninclude, but are not limited to, promoter sequences, transcriptioninitiation sequences, and enhancer sequences.

Variant FVIII proteins, produced by gene expression in a recombinantprokaryotic or eukaryotic system may be purified according to methodsknown in the art. In a preferred embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinant proteinis expressed and thereafter secreted from the host cell, to be easilypurified from the surrounding medium. If expression/secretion vectorsare not used, an alternative approach involves purifying the recombinantprotein by affinity separation, such as by immunological interactionwith antibodies that bind specifically to the recombinant protein ornickel columns for isolation of recombinant proteins tagged with 6-8histidine residues at their N-terminus or C-terminus. Alternative tagsmay comprise the FLAG epitope, GST or the hemagglutinin epitope. Suchmethods are commonly used by skilled practitioners.

FVIII proteins, prepared by the aforementioned methods, may be analyzedaccording to standard procedures. For example, such proteins may beassessed for altered coagulation properties according to known methods.

As discussed above, a convenient way of producing a polypeptideaccording to the present invention is to express nucleic acid encodingit, by use of the nucleic acid in an expression system. A variety ofexpression systems of utility for the methods of the present inventionare well known to those of skill in the art.

Accordingly, the present invention also encompasses a method of making apolypeptide (as disclosed), the method including expression from nucleicacid encoding the polypeptide (generally nucleic acid). This mayconveniently be achieved by culturing a host cell, containing such avector, under appropriate conditions which cause or allow production ofthe polypeptide. Polypeptides may also be produced in in vitro systems.

III. Uses of FVIII Proteins and FVIII-Encoding Nucleic Acids

Variant FVIII nucleic acids encoding polypeptides having alteredcoagulation activities may be used according to this invention, forexample, as therapeutic and/or prophylactic agents (protein or nucleicacid) which modulate the blood coagulation cascade or as a transgene ingene-, and or cell-based strategies for continuous expression of FVIIIand its variants in hemophilia A patients. The present inventors havediscovered modifications of FVIII molecules which result in increasedcoagulation activity and greater stability thereby effectively improvinghemostasis.

A. Variant FVIII Polypeptides

In a preferred embodiment of the present invention, variant FVIIIpolypeptides may be administered to a patient via infusion in abiologically compatible carrier, preferably via intravenous injection.The variant FVIIIs of the invention may optionally be encapsulated intoliposomes or mixed with other phospholipids or micelles to increasestability of the molecule. FVIII may be administered alone or incombination with other agents known to modulate hemostasis (e.g., FactorV, Factor Va or derivatives thereof). An appropriate composition inwhich to deliver FVIII polypeptides may be determined by a medicalpractitioner upon consideration of a variety of physiological variables,including, but not limited to, the patient's condition and hemodynamicstate. A variety of compositions well suited for different applicationsand routes of administration are well known in the art and are describedhereinbelow.

The preparation containing the purified FVIII analog contains aphysiologically acceptable matrix and is preferably formulated as apharmaceutical preparation. The preparation can be formulated usingsubstantially known prior art methods, it can be mixed with a buffercontaining salts, such as NaCl, CaCl₂, and amino acids, such as glycineand/or lysine, and in a pH range from 6 to 8. Until needed, the purifiedpreparation containing the FVIII analog can be stored in the form of afinished solution or in lyophilized or deep-frozen form. Preferably thepreparation is stored in lyophilized form and is dissolved into avisually clear solution using an appropriate reconstitution solution.

Alternatively, the preparation according to the present invention canalso be made available as a liquid preparation or as a liquid that isdeep-frozen.

The preparation according to the present invention is especially stable,i.e., it can be allowed to stand in dissolved form for a prolonged timeprior to application.

The preparation according to the present invention can be made availableas a pharmaceutical preparation with FVIII activity in the form of aone-component preparation or in combination with other factors in theform of a multi-component preparation. Prior to processing the purifiedprotein into a pharmaceutical preparation, the purified protein issubjected to the conventional quality controls and fashioned into atherapeutic form of presentation. In particular, during the recombinantmanufacture, the purified preparation is tested for the absence ofcellular nucleic acids as well as nucleic acids that are derived fromthe expression vector, preferably using a method, such as is describedin EP 0 714 987.

The pharmaceutical protein preparation may be used at dosages of between30-100 IU/kg (One I.U is 100 ng/ml) at as single daily injection or upto 3 times/day for several days. Patients may be treated immediatelyupon presentation at the clinic with a bleed. Alternatively, patientsmay receive a bolus infusion every eight to twelve hours, or ifsufficient improvement is observed, a once daily infusion of the variantFVIII described herein.

B. FVIII-Encoding Nucleic Acids

FVIII-encoding nucleic acids may be used for a variety of purposes inaccordance with the present invention. In a preferred embodiment of theinvention, a nucleic acid delivery vehicle (i.e., an expression vector)for modulating blood coagulation is provided wherein the expressionvector comprises a nucleic acid sequence coding for a variant FVIIIpolypeptide, or a functional fragment thereof as described herein.Administration of FVIII-encoding expression vectors to a patient resultsin the expression of FVIII polypeptide which serves to alter thecoagulation cascade. In accordance with the present invention, an FVIIIencoding nucleic acid sequence may encode an FVIII polypeptide asdescribed herein whose expression increases hemostasis. In a preferredembodiment, a FVIII nucleic acid sequence encodes a human FVIIIpolypeptide variant.

Expression vectors comprising variant FVIII nucleic acid sequences maybe administered alone, or in combination with other molecules useful formodulating hemostasis. According to the present invention, theexpression vectors or combination of therapeutic agents may beadministered to the patient alone or in a pharmaceutically acceptable orbiologically compatible compositions.

In a preferred embodiment of the invention, the expression vectorcomprising nucleic acid sequences encoding the variant FVIII variants isa viral vector. Viral vectors which may be used in the present inventioninclude, but are not limited to, adenoviral vectors (with or withouttissue specific promoters/enhancers), adeno-associated virus (AAV)vectors of multiple serotypes (e.g., AAV-1 to AAV-12, and others) andhybrid AAV vectors, lentivirus vectors and pseudo-typed lentivirusvectors [e.g., Ebola virus, vesicular stomatitis virus (VSV), and felineimmunodeficiency virus (FIV)], herpes simplex virus vectors, vacciniavirus vectors, retroviral vectors, lentiviral vectors, non-viral vectorsand others.

In a preferred embodiment of the present invention, methods are providedfor the administration of a viral vector comprising nucleic acidsequences encoding a variant FVIII, or a functional fragment thereof.AAV vectors and lentiviral vectors have broad utility in the methods ofthe present invention and preferably do not include any viral genesassociated with pathogenesis. Most preferably, only the essential partsof vector e.g., the ITR and LTR elements, respectively are included.Direct delivery of vectors or ex-vivo transduction of human cells andfollowed by infusion into the body will result in expression of variantFVIIIs thereby exerting a beneficial therapeutic effect on hemostasis.In the context of the variant Factor VIII described herein, suchadministration enhances pro-coagulation activity.

Recombinant AAV and lentiviral vectors have found broad utility for avariety of gene therapy applications. Their utility for suchapplications is due largely to the high efficiency of in vivo genetransfer achieved in a variety of organ contexts.

AAV and lentiviral particles may be used to advantage as vehicles foreffective gene delivery. Such virions possess a number of desirablefeatures for such applications, including tropism for dividing andnon-dividing cells. Early clinical experience with these vectors alsodemonstrated no sustained toxicity and immune responses were minimal orundetectable. AAV are known to infect a wide variety of cell types invivo and in vitro by receptor-mediated endocytosis or by transcytosis.These vector systems have been tested in humans targeting retinalepithelium, liver, skeletal muscle, airways, brain, joints andhematopoietic stem cells. It is likely that non-viral vectors based onplasmid DNA or minicircles will be also suitable gene transfer vectorsfor a large gene as that encoding FVIII.

It is desirable to introduce a vector that can provide, for example,multiple copies of a desired gene and hence greater amounts of theproduct of that gene. Improved AAV and lentiviral vectors and methodsfor producing these vectors have been described in detail in a number ofreferences, patents, and patent applications, including: Wright J. F.(Hum Gene Ther 20:698-706, 2009) which is the technology used for theproduction of clinical grade vector at our facility at Children'sHospital of Philadelphia. Lentiviral vector can be produced at CHOP andthe other vectors are available through the Lentivirus vector productioncore laboratory by NHLBI Gene Therapy Resource Program (GTRP)—LentivirusVector Production Core Laboratory. For some applications, an expressionconstruct may further comprise regulatory elements which serve to driveexpression in a particular cell or tissue type. Such regulatory elementsare known to those of skill in the art and discussed in depth inSambrook et al. (1989) and Ausubel et al. (1992). The incorporation oftissue specific regulatory elements in the expression constructs of thepresent invention provides for at least partial tissue tropism for theexpression of the variant FVIIIs or functional fragments thereof. Forexample, nucleic acid sequences encoding variant FVIII under the controlof a cytomegalovirus (CMV) promoter can be employed for skeletal muscleexpression or the hAAT-ApoE and others for liver specific expression.Hematopoietic specific promoters in lentiviral vectors may also be usedto advantage in the methods of the present invention.

Exemplary Methods for Producing AAV Vectors

AAV for recombinant gene expression have been produced in the humanembryonic kidney cell line 293 and extensively recently reviewed by theDirector of Clinical Vector Core at CHOP, Dr. J. F. Wright (Hum GeneTher 20:698-706, 2009). Briefly, AAV vectors are engineered fromwild-type AAV, a single-stranded DNA virus that is non-pathogenic. Theparent virus is non-pathogenic, the vectors have a broad host range, andthey can infect both dividing and non-dividing cells. The vector isengineered from the virus by deleting the rep and cap genes andreplacing these with the transgene of interest under the control of aspecific promoter. For recombinant AAV preparation, the upper size limitof the sequence that can be inserted between the two ITRs is ˜5.0 kb.The plasmids expressing canine or human FIX under the control of the CMVpromoter/enhancer and a second plasmid supplying adenovirus helperfunctions along with a third plasmid containing the AAV-2 rep and capgenes were used to produce AAV-2 vectors, while a plasmid containingeither AAV-1, AAV-6, or AAV-8 cap genes and AAV-2 rep gene and ITR's areused to produce the respective alternate serotype vectors (Gao et al.,(2002) Proc. Natl Acad. Sci. USA 99:11854-11859; Xiao et al., (1999) J.Virol. 73:3994-4003; Arruda et al., (2004) Blood 103:85-92). AAV vectorsare purified by repeated CsCl density gradient centrifugation and thetiter of purified vectors determined by quantitative dot-blothybridization. Vectors used for experiments in dogs and mice presentedherein were prepared by the Vector Core at The Children's Hospital ofPhiladelphia.

Also included in the present invention is a method for modulatinghemostasis comprising providing cells of an individual with a nucleicacid delivery vehicle encoding a variant FVIII polypeptide and allowingthe cells to grow under conditions wherein the FVIII polypeptide isexpressed.

From the foregoing discussion, it can be seen that FVIII polypeptides,and FVIII polypeptide expressing nucleic acid vectors may be used in thetreatment of disorders associated with aberrant blood coagulation.

C. Pharmaceutical Compositions

The expression vectors of the present invention may be incorporated intopharmaceutical compositions that may be delivered to a subject, so as toallow production of a biologically active protein (e.g., a variant FVIIIpolypeptide or functional fragment or derivative thereof) or by inducingcontinuous expression of the FVIII transgene in vivo by gene- and orcell-based therapies or by ex-vivo modification of the patient's ordonor's cells. In a particular embodiment of the present invention,pharmaceutical compositions comprising sufficient genetic material toenable a recipient to produce a therapeutically effective amount of avariant FVIII polypeptide can influence hemostasis in the subject.Alternatively, as discussed above, an effective amount of the variantFactor VIII polypeptide may be directly infused into a patient in needthereof. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents (e.g., co-factors) which influencehemostasis.

In preferred embodiments, the pharmaceutical compositions also contain apharmaceutically acceptable excipient. Such excipients include anypharmaceutical agent that does not itself induce an immune responseharmful to the individual receiving the composition, and which may beadministered without undue toxicity. Pharmaceutically acceptableexcipients include, but are not limited to, liquids such as water,saline, glycerol, sugars and ethanol. Pharmaceutically acceptable saltscan also be included therein, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., 18th Edition, Easton, Pa.[1990]).

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding, free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they may be placedin an appropriate container and labeled for treatment. Foradministration of FVIII-containing vectors or polypeptides, suchlabeling would include amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended therapeutic purpose.Determining a therapeutically effective dose is well within thecapability of a skilled medical practitioner using the techniques andguidance provided in the present invention. Therapeutic doses willdepend on, among other factors, the age and general condition of thesubject, the severity of the aberrant blood coagulation phenotype, andthe strength of the control sequences regulating the expression levelsof the variant FVIII polypeptide. Thus, a therapeutically effectiveamount in humans will fall in a relatively broad range that may bedetermined by a medical practitioner based on the response of anindividual patient to vector-based FVIII treatment.

D. Administration

The variant Factor VIII polypeptides, alone or in combination with otheragents may be directly infused into a patient in an appropriatebiological carrier as described hereinabove. Expression vectors of thepresent invention comprising nucleic acid sequences encoding variantFVIII, or functional fragments thereof, may be administered to a patientby a variety of means (see below) to achieve and maintain aprophylactically and/or therapeutically effective level of the FVIIIpolypeptide. One of skill in the art could readily determine specificprotocols for using the FVIII encoding expression vectors of the presentinvention for the therapeutic treatment of a particular patient.Protocols for the generation of adenoviral vectors and administration topatients have been described in U.S. Pat. Nos. 5,998,205; 6,228,646;6,093,699; 6,100,242; and International Patent Application Nos. WO94/17810 and WO 94/23744, which are incorporated herein by reference intheir entirety.

Variant FVIII encoding adenoviral vectors of the present invention maybe administered to a patient by any means known. Direct delivery of thepharmaceutical compositions in vivo may generally be accomplished viainjection using a conventional syringe, although other delivery methodssuch as convection-enhanced delivery are envisioned (See e.g., U.S. Pat.No. 5,720,720). In this regard, the compositions may be deliveredsubcutaneously, epidermally, intradermally, intrathecally,intraorbitally, intramucosally, intraperitoneally, intravenously,intraarterially, orally, intrahepatically or intramuscularly. Othermodes of administration include oral and pulmonary administration,suppositories, and transdermal applications. A clinician specializing inthe treatment of patients with blood coagulation disorders may determinethe optimal route for administration of the adenoviral vectorscomprising FVIII nucleic acid sequences based on a number of criteria,including, but not limited to: the condition of the patient and thepurpose of the treatment (e.g., enhanced or reduced blood coagulation).

The present invention also encompasses AAV vectors comprising a nucleicacid sequence encoding a variant FVIII polypeptide.

Also provided are lentivirus or pseudo-typed lentivirus vectorscomprising a nucleic acid sequence encoding a variant FVIII polypeptide.

Also encompassed are naked plasmid or expression vectors comprising anucleic acid sequence encoding a variant FVIII polypeptide.

The following materials and methods are provided to facilitate thepractice of Example I.

Purification of Canine and Human FVIII-BDD:

Plasmids encoding hFVIII-BDD or cFVIII-BDD were introduced into babyhamster kidney (BHK) cells and high producing stable clones wereestablished as described using standard techniques (Toso et al. (2004)J. Biol. Chem. 279:21643-21650). Cells were expanded into triple flasksand cultured in DMEM/F12 media (no phenol red) supplemented with ITS,2.5 mM CaCl₂ and 1.0 mg/mL Albumax (Invitrogen, Carlsbad, Calif.).Variants of FVIII containing amino acid substitutions were generatedusing site directed mutagenesis. Conditioned media was collected dailyfor 4-6 days, centrifuged, and inhibitors added (10 μM APMSF and 1 mMbenzamidine). For purification, media was processed daily and loadedonto a ˜70 mL SPSepharose FF column (Amersham Biosciences, Piscataway,N.J.) equilibrated in 20 mM MES, 0.15 M NaCl, 5 mM CaCl₂, 0.01%Tween-80, pH 6.8. The column was washed with the same buffer and elutedwith 20 mM MES, 0.65 M NaCl, 5 mM CaCl₂, 0.01% Tween-80, pH 6.8.Fractions containing cFVIII-BDD (monitored by clotting assay) werestored at −80° C. Following successive daily runs of the SP-Sepharosecolumn, all fractions containing activity were pooled and diluted with20 mM MES/5 mM CaCl2/0.01% Tween-80, pH 6.8 and then loaded on a PorosHS/20 column (10×100 mm; Applied Biosystems, Foster City, Calif.)equilibrated with the same buffer. The column was washed with 20 mM MES,2 mM CaCl₂, pH 6.0 and then eluted with a 0-1.0 M NaCl gradient.cFVIII-BDD containing fractions were pooled, and then and diluted with20 mM HEPES/5 mM CaCl2, pH 7.4 and then loaded on a Poros HQ/20 column(4.6×100 mm; Applied Biosystems, Foster City, Calif.) equilibrated withthe same buffer. The column was washed with 20 mM HEPES/5 mM CaCl2, pH7.4 and then eluted with a 0-65 M NaCl gradient. Fractions containingactivity were dialyzed versus 20 mM Hepes, 2 mM CaCl2, pH 7.4 for 2 hrand the protein was stored at −80° C. in small aliquots.

Protein specific activity was determined by activated partialthromboplastin time (aPTT) with minor modifications (12). Decay ofactivated FVIII activity was monitored by purified component assay usingboth reconstituted human factor Xase complex and plasma models aspreviously described (11). N-terminal sequencing was determined in thelaboratory of Dr. Alexander Kurosky and Dr. Steven Smith at UTMB(Galveston, Tex.). Enzymatic cleavage of N-linked glycans was carriedout using recombinant N-glycosidase F (Boehringer Mannheim,Indianapolis, Ind.) as reported before (13).

cFVIII-BDD Antigen ELISA

cFVIII-BDD protein was used for the generation of a series of rabbitanti-cFVIII-BDD polyclonal and murine monoclonal anticFVIII-BDDantibodies (Green Mountain Antibodies, Burlington, Vt.) (See FIG. 1).Anti-cFVIII antibodies were detected by Bethesda assay (14) or bycFVIII-specific IgG antibodies by ELISA. A monoclonal antibody to thelight chain (clone 2C4.1C3) or heavy chain (clone 4B1.2C8) to capturethe protein (2 μg/mL) followed by a rabbit anti-cFVIII-BDD polyclonal asa secondary antibody (2 μg/mL). The cFVIII-BDD was detected with a goatanti-rabbit antibody conjugated to horseradish peroxidase at a dilutionof 1:15,000 (Jackson ImmunoResearch Laboratories, Inc., West Grove,Pa.). The standard curve was generated using serial dilution ofrecombinant cFVIII-BDD. Half-life and recovery were calculated aspreviously described (15, 16).

Anti-cFVIII Specific IgG ELISA

ELISA was used to detect cFVIII-specific IgG antibodies by usingpurified cFVIII-BDD protein (1 μg/mL) to capture IgG1 or IgG2 antibodiesin dog serum. Canine reference serum (B ethyl Laboratories Inc.,Montgomery, Tex.) with known concentrations of IgG1 and IgG2 was used asa standard by coating with serial dilutions of the canine serum. Canineserum samples were diluted in LowCross-Buffer (Candor Bioscience GmbH,Germany). The IgG was detected with goat anti-canine IgG1 conjugated tohorseradish peroxidase or sheep anti-canine IgG2 conjugated tohorseradish peroxidase (Bethyl Laboratories Inc., Montgomery, Tex.)diluted 1:1000 in LowCross-Buffer (Candor, Bioscience GmbH,Weissensberg, Germany).

The following examples are provided to illustrate certain embodiments ofthe invention. They are not intended to limit the invention in any way.

Example 1 Recombinant Canine B-Domain Deleted FVIII Exhibits HighSpecific Activity and is Safe in the Canine Hemophilia A Model

Using identical expression systems, we found that cFVIII-BDD typicallyyields 0.5 mg/L which is 3-fold higher than hFVIII-BDD (0.16 mg/L).Notably, purified cFVIII-BDD existed predominantly as a single-chainprotein (>75% of total) whereas, as expected, hFVIII-BDD was primarily aheterodimer (FIG. 2A, B). The amino acid recognition sequence for thePACE/furin in cFVIII (HHQR) (SEQ ID NO:1) (17) differs from human andporcine FVIII (RHQR) (SEQ ID NO:2) and may explain the predominantsingle-chain form of cFVIII (17). Following treatment with thrombincFVIII-BDD was properly activated and no major differences were observedbetween hFVIII-BDD and cFVIII-BDD (FIG. 2B). The Al domain migrationpattern differs between both FVIII species. However, removal of N-linkedglycan resulted in similar migration of the Al domains (data not shown).These data indicate that either the glycosylation structure on the Aldomains is different or that possibly only one site in the human Aldomain is glycosylated. Moreover, N-terminal sequencing of relevant bandyields the expected results (data not shown). Using a one-stage aPTT,the specific activity of cFVIII-BDD (33,926±675 U/mg) was ˜3-fold higherthan hFVIII-BDD (12,345±787 U/mg) (p<0.001). Similar findings wereobtained after thrombin activation of canine and human FVIII in thetwo-stage aPTT (756,754±60,592 vs. 343,066±2090 U/mg, p<0.003) yieldingan activation quotient (AQ) of 28 and 22 for human and canine,respectively (FIG. 2, panel C). Typically, low AQ representscontamination with activated forms of the protein and results in falsehigh protein activity. These findings were consistent using threeseparate cFVIII-BDD preparations. Taken together these data usingpurified FVIII protein support the conclusions that cFVIII has anelevated intrinsic specific activity.

Following activation, FVIIIa rapidly loses activity due to A2-domaindissociation from the A1/A2/A3-C1-C2 heterotrimer. Purified cFVIII-BDDor hFVIII-BDD were rapidly activated (˜30 s) with thrombin and residualcofactor activity was monitored over time. Using either a purifiedcomponent assay (FIG. 2, panel D) or clotting assay (data not shown) wefound that the half-life of cFVIII was 3-fold longer than hFVIII. Thesefindings suggest that cFVIIIa exhibits increased affinity for theA2-domain compared to hFVIIIa. While these data could, in part, accountfor the high specific activity of cFVIII, both porcine (17) and murineFVIIIBDD (11) also have enhanced A2-domain stability compared to hFVIIIbut apparently have equivalent specific activity to hFVIII. Thus, it ispossible that the increased specific activity of cFVIII is due thesingle chain protein with higher stability.

To test the efficacy and safety of the cFVIII-BDD, we injected a seriesof adult and neonate HA dogs. In these dogs, no circulating FVIIIantigen was detected, which is consistent with humans with the analogousFVIII mutation. In normal dogs cFVIII levels are 80-130 ng/mL which iscomparable to human levels (100-200 ng/mL) and to cFVIII levelspreviously described (18).

HA dogs received cFVIII-BDD at doses of 2.5 μg/kg every 30 days for 2-4months and serial plasma samples were collected through four weeks aftereach protein injection. cFVIIIBDD was functional as evidenced by theshortening of the whole blood clotting time (WBCT) and increased cFVIIIclotting activity (FIG. 3). The recovery of the protein measured at 5-10minutes (n=5 infusions) post-injection was excellent, reaching levels of71.8%±9.2%. There was a good correlation between cFVIII activity andantigen levels (FIG. 3B). The levels of cFVIII slowly declined after theinfusion and returned to baseline within 48-56 hours with a calculatedhalf-life of 12-14 hours. There was no local or systemic toxicity and noevidence of pathological activation of coagulation. Together these datademonstrate that cFVIII-BDD is safe and efficacious in inducingsustained hemostasis in vivo and has a protein half-life comparable tothe pharmacokinetics of hFVIII-BDD in HA dogs and from clinicalexperience in humans (19).

The use of these outbred immunocompetent HA dogs provide an ideal modelto test the immunogenicity of cFVIII-BDD protein in both naïve neonatesand adult dogs previously exposed to plasma-derived cFVIII. These dogsdo not develop antibody to cFVIII upon infusion of plasma-derivedcFVIII. Here, in adult dogs, no antibodies to cFVIII-BDD were detectedby Bethesda assay or cFVIII-specific IgGs after repetitive exposure tothe protein (FIG. 3C). Furthermore, neonate dogs (n=3) exclusivelyexposed to cFVIII-BDD or small amounts of plasma-derived FVIII (n=2)also did not develop antibodies to cFVIII. In an HA dog with aninhibitor to plasma-derived cFVIII, inhibitor titers of 4 B.U.corresponds to 3000-4000 ng/mL of IgG2. These data are in contrast tothe strong immune responses of adult HA dogs to hFVIII characterized bylong-lasting antibody to hFVIII after exposure to the protein orfollowing delivery of hFVIII gene or cell-based therapies (1, 16, 19,20). Thus, cFVIII-BDD presents no immunogenicity in this pivotal HA dogmodel which is essential for determining long-term efficacy and safetyof novel therapeutic strategies for HA.

We sought to compare the rates of inactivation of cFVIII-BDD withhFVIII-BDD in human plasma containing inhibitors to FVIII. The recoveryof cFVIII after incubation with inhibitors was 40-45% higher than hFVIII(See FIG. 4). The higher survival of cFVIII in the presence of humaninhibitors further supports the investigation of cFVIII as a potentialbypass strategy for hemophilia.

The recombinant expression of cFVIII-BDD allowed us to generate largeamounts of protein (>20 mg), develop valuable antibodies and begin tounravel intrinsic properties of the protein that may impact thedevelopment of treatment of hemophilia. The enhanced biological activityof cFVIII could partially result from the secretion of cFVIII as asingle-chain protein and hFVIII variants with canine PACE/furin cleavagesites may help define whether these modifications would improveproduction and stability of the recombinant protein. Furthermore adetailed analysis of Xase complex assembly in kinetic characterizationwith canine FVIIIa will shed light on its apparent increased specificactivity compared to hFVIII.

The efficacy and safety data from studies on non-inhibitor prone HA dogsdemonstrate that cFVIII-BDD is an attractive option for the treatment ofbleeds and for prophylaxis in dogs during complex or invasiveprocedures. The ability to detect non-neutralizing IgG antibodies inaddition to neutralizing antibodies provides the opportunity toelucidate conflicting findings in gene or cell-based therapy in thesedogs (21-24). A more comprehensive phenotypic characterization of the HAdogs is now feasible and further improves the relevance of preclinicalstudies for a new generation of gene- and cell-based therapies forhemophilia.

Example 2 Generation of Mutant Human and Canine FVIII with ModifiedPACE-Furin Cleavage Sites

Using the same techniques described above, we generated human BDD-FVIIIwith the WT and mutant R1645H proteins. The purified products were runon a SDS PAGE. The canine FVIII amino acid recognition sequence forintracellular cleavage by PACE/fuin (HHQR) (SEQ ID NO:1) differs fromthat of human and porcine FVIII (RHQR) (SEQ ID NO:2). We tested thepossible role of this cleavage site in increased cFVIII single chainstability and activity. Single substitutions of R→H in human FVIII andH→R at a position homologous in canine FVIII resulted in a shift of ˜2-3fold in the ratio of single chain to cleaved FVIII in the secretedmaterial in the anticipated direction. See FIG. 5. Moreover, theactivated HFVIII^(R1645H) had a 3-fold increase in half-life compared toWT hBFVIII. See FIG. 6. Notably, in FIG. 6, the A2 domain dissociationof mutant human FVIII R1645H is similar to that of the wild-type cFVIIIand porcine FVIII. These studies suggest a single amino acidsubstitution at position 1645 enhances human FVIII biological functionactivity.

Effects of Human FVIII R1650H Variant at Both Micro andMacrocirculation.

As discussed above, canine FVIII has greater activity, in part due toits increased stability as it is expressed as predominantly as a singlechain, likely involving a single R1650H (RH) substitution at thePACE/Furin site in cFVIII. Lenti/BMT pFVIII studies expressingphBFVIIIRH showed that the FVIII variant containing this amino acidsubstitution and B domain deletion was expressed at levels in micecomparable to those observed in transgenic hemophilia A mice expressingwild-type human FVIII. Moreover, the FVIII variant was more efficaciousin several bleeding models, including near-normal hemostais in thecremaster laser injury model (microcirculation) or carotid artery model(macrocirculation) in hemophilia A recipient mice. See FIG. 7. This isfirst lenti/BMT pFVIII-expressing HA mouse with near-normal hemostasis.Preliminary megakaryocyte counts and apoptosis studies show thatphBFVIIIRH is not deleterious to megakaryocytes as observed by otherswhen wild-type FVIII was expressed in these cells. These studies provideimportant new insights into pF8 efficacy for the treatment of hemophiliaA.

The hFVIII 1645H variant exhibits higher stability due to the slowdissociation of the A2-domain. Accordingly, this variant may be used toadvantage upon tissue injury in order to enhance the generation andduration of clot formation thereby providing a more efficienthemostasis. Additional alterations of the PACE-furin cleavage siteshould yield similar resistant FVIII variants. Such alterations include,without limitation, deletion of one or more amino acids within thecleavage site, and substitution of the R in the human sequence with anamino acid such as, serine, lysine, methionine, cysteine, proline andtyrosine. Secondly, any of these variants will useful for combining withother forms of FVIII, including, without limitation, IR8 that isresistant to the inactivation by activated protein C. This combinationof the hFVIII 1645 (or other PACE-furin resistance forms) with the IR8variant may further enhance the efficacy of the FVIII in inducinghemostasis. Finally, the protein product described herein could alsoused for site specific pegylation aimed at increasing the proteinhalf-life or encapsulated with various compounds to enhance efficacywhile maintaining appropriate safety parameters.

As discussed above, data on expression of human FVIII-RH mutation in theliver of hemophilia A mice demonstrate that at the same vector dose,using adeno-associated viral vector (AAV, FIG. 8) for liver-restrictedexpression resulted in 50% higher FVIII expression levels compared tothe wild-type human FVIII (FVIII-WT), suggesting that this FVIII varianthas a higher stability in the plasma (FIG. 9)

In subsequent experiments, animals expressing FVIII-RH or FVIII-WTunderwent to hemostatic challenges and the most informative data is thereduced blood loss to levels compared to hemostastic normal mice. Thefirst time we observed such normal correction (FIG. 10).

In a series of hemostatic challenges at the macrocirculation (carotidartery, FIG. 11) and at microcirculation (cremaster arteriole, ongoingexperiments), FVIII-RH proved functional to prevent bleeding at thesesites.

Experiments with purified proteins injected exogenously, were associatedwith formation of stable clotting factor when FVIII-RH was compared toFVIII-WT with minimal dissolution of the clot (as commonly observed withFVIII-WT).

In a different experimental system; FVIII-RH was expressed specificallyin the platelet and its efficacy tested in the cremaster arterioleinjury model (FIG. 12A). Two unexpected observations were finding: (1)FVIII-RH resulted in stable clot (unpublished), as observed in thetransgenic mice with the highest expression levels (FIG. 12B) (see Blood2011 for the FVIII-WT and not for the variant RH), (2) the accumulationof fibrin in this model, surpassed that of hemostatic normal mice, whichsuggest that local increase of FVIII-RH upon platelet activation has apotent hemostatic effect (FIG. 12C).

In summary, using distinct models of expression of FVIII-RH in theplasma (derived from liver by AAV vector) or by injection of purifiedprotein, provide further support for the use of this variant in clinics.Secondly, expression of FVIII-RH in platelet provides strong evidencethat local increment of the protein has a potent procoagulant effect.Accordingly, the best performing FVIII variant(s) can be used for (a)protein production for the treatment of bleeding, either prophylaticallyor in response to a bleed, (b) in transgenes for direct gene delivery byviral or non-viral vectors to the liver, skeletal muscle, or skin. Inaddition, these vectors and lentiviral, retroviral vectors can beemployed to target hematopoietic stem cell for expression in cells fromthe bone marrow. They can also be utilized to drive expression ofvariant FVIII in induced Progenitor cells (iPS) or human or non-humanembryonic stem cells. Such cells can be transduced ex vivo and thenreturned to the patient via IV or local injection. In one approach, thecells are derived from the patient. In another, the cells may beobtained from an immunologically compatible donor.

REFERENCES

-   1. Nichols T C, Dillow A M, Franck H W, et al. Protein replacement    therapy and gene transfer in canine models of hemophilia A,    hemophilia B, von Willebrand disease, and factor VII deficiency.    Ilar J. 2009; 50:144-167.-   2. Lakich D, Kazazian H H, Antonarakis S E, Gitschier J. Inversions    disrupting the factor VIII gene as a common cause of severe    haemophilia A. Nature Genet. 1993; 5:236-241.-   3. Graham J B, Buckwalter J A, Hartley L J, Brinkhous K M. Canine    hemophilia: Observations on the course, the clotting anomaly, and    the effects of blood transfusion. J Exp Med. 1949; 90:97-102.-   4. Lozier J N, Dutra A, Pak E, et al. The Chapel Hill hemophilia A    dog colony exhibits a factor VIII gene inversion. Proc Natl Acad Sci    USA. 2002; 99:12991-12996.-   5. Cameron C, Notley C, Hoyle S, et al. The canine factor VIII cDNA    and 5′ flanking sequence. Thromb Haemost. 1998; 79:317-322.-   6. Viel K R, Ameri A, Abshire T C, et al. Inhibitors of factor VIII    in black patients with hemophilia. N Engl J Med. 2009;    360:1618-1627.-   7. Sarkar R, Tetreault R, Gao G, et al. Total correction of    hemophilia A mice with canine FVIII using an AAV 8 serotype. Blood.    2004; 103:1253-1260.-   8. Cao W, Krishnaswamy S, Camire R M, Lenting P J, Zheng X L. Factor    VIII accelerates proteolytic cleavage of von Willebrand factor by    ADAMTS13. Proc Natl Acad Sci USA. 2008; 105:7416-7421.-   9. Kaufman R J, Davies M V, Wasley L C, Michnick D. Improved vectors    for stable expression of foreign genes in mammalian cells by use of    the untranslated leader sequence from EMC virus. Nucleic Acids Res.    1991; 19:4485-4490.-   10. Toso R, Camire R M. Removal of B-domain sequences from factor V    rather than specific proteolysis underlies the mechanism by which    cofactor function is realized. J Biol Chem. 2004; 279:21643-21650.-   11. Doering C, Parker E T, Healey J F, Craddock H N, Barrow R T,    Lollar P. Expression and characterization of recombinant murine    factor VIII. Thromb Haemost. 2002; 88:450-458.-   12. Lollar P, Parker E T, Fay P J. Coagulant properties of hybrid    human/porcine factor VIII molecules. J Biol Chem. 1992;    267:23652-23657.-   13. Arruda V R, Hagstrom J N, Deitch J, et al. Posttranslational    modifications of recombinant myotube-synthesized human factor IX.    Blood. 2001; 97:130-138.-   14. Herzog R W, Mount J D, Arruda V R, High K A, Lothrop C D, Jr.    Muscle-directed gene transfer and transient immune suppression    result in sustained partial correction of canine hemophilia B caused    by a null mutation. Mol Ther. 2001; 4:192-200.-   15. Brinkhous K M, Sandberg H, Garris J B, et al. Purified human    factor VIII procoagulant protein: comparative hemostatic response    after infusions into hemophilic and von Willebrand disease dogs.    Proc Natl Acad Sci USA. 1985; 82:8752-8756.-   16. Brinkhous K M, Hedner U, Garris J B, Diness V, Read M S. Effect    of recombinant factor VIIa on the hemostatic defect in dogs with    hemophilia A, hemophilia B, and von Willebrand disease. Proc Natl    Acad Sci USA. 1989; 86:1382-1386.-   17. Doering C B, Healey J F, Parker E T, Barrow R T, Lollar P. High    level expression of recombinant porcine coagulation factor VIII. J    Biol Chem. 2002; 277:38345-38349.-   18. Shibata M, Rowle F, Labelle A, et al. Characterization of    recombinant canine FVIII and quantitative determination of factor    FVIII in canine plasma. J Thromb Haemost. 2005; 3:P0033.-   19. Brinkhous K, Sandberg H, Widlund L, et al. Preclinical    pharmacology of albumin-free Bdomain deleted recombinant    factor VIII. Semin Thromb Hemost. 2002; 28:269-272.-   20. Connelly S, Mount J, Mauser A, et al. Complete short-term    correction of canine hemophilia A by in vivo gene therapy. Blood.    1996; 88:3846-3853.-   21. Brown B D, Shi C X, Powell S, Hurlbut D, Graham F L,    Lillicrap D. Helper-dependent adenoviral vectors mediate therapeutic    factor VIII expression for several months with minimal accompanying    toxicity in a canine model of severe hemophilia A. Blood. 2004;    103:804-810.-   22. Chuah M K, Schiedner G, Thorrez L, et al. Therapeutic factor    VIII levels and negligible toxicity in mouse and dog models of    hemophilia A following gene therapy with high-capacity adenoviral    vectors. Blood. 2003; 101:1734-1743.-   23. Jiang H, Lillicrap D, Patarroyo-White S, et al. Multiyear    therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor    VIII to hemophilia A mice and dogs. Blood. 2006; 108:107-115.-   24. McCormack W M, Jr., Seiler M P, Bertin T K, et al.    Helper-dependent adenoviral gene therapy mediates long-term    correction of the clotting defect in the canine hemophilia A model.    J Thromb Haemost. 2006; 4:1218-1225.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. An isolated recombinant nucleic acid encoding aFactor VIII (FVIII) variant having a modified PACE/furin cleavage siteand a B domain deletion (BDD) for modulating hemostasis, said PACE/furincleavage site consisting of amino acids RHQR (SEQ ID NO:2), said FVIIIvariant being a variant selected from the group consisting essentiallyof: i) a FVIII variant with said BDD and wherein R is substituted withan H in the PACE/furin cleavage site; ii) a FVIII variant with said BDDand wherein H is substituted with a S in the PACE/furin cleavage site;iii) a FVIII variant with said BDD and wherein one amino acid at saidPACE/furin cleavage site is deleted; iv) a FVIII variant with said BDDand wherein R at said PACE/furin cleavage site is substituted with anamino acid selected from the group consisting of a seine, lysine,methionine, cysteine, proline and tyrosine; and v) a FVIII variantwherein one or more amino acids at said PACE/furin cleavage site and a Bdomain are deleted, each of i), ii), iii), iv) and v) exhibitingincreased specific activity and stability relative to FVIII-BDD lackingsaid substitution and deletions.
 2. An expression vector comprising thenucleic acid of claim
 1. 3. The vector of claim 2, selected from thegroup consisting of an adenoviral vector, an adenovirus-associated virus(AAV) vector, a retroviral vector, a plasmid, and a lentiviral vector.4. A method for treatment of a hemostasis related disorder in a patientin need thereof comprising administration of a therapeutically effectiveamount of the vector of claim 3 in a biologically acceptable carrier,wherein said vector is an AAV vector.
 5. The method of claim 4, whereinsaid FVIII variant is a pro-coagulant and said disorder is selected fromthe group consisting of hemophilia A, von Willebrand diseases andbleeding associated with trauma, injury, thrombosis, thrombocytopenia,stroke, coagulopathy, disseminated intravascular coagulation (DIC) andover-anticoagulation treatment disorders.
 6. The method of claim 4 orclaim 5, wherein said FVIII variant is encapsulated in a liposome ormixed with phospholipids or micelles.
 7. A pharmaceutical compositioncomprising the adenoviral vector, adenovirus-associated virus (AAV)vector, retroviral vector, plasmid, or lentiviral vector of claim 3 in abiologically compatible carrier.
 8. A host cell expressing the FVIIIvariant encoded by the nucleic acid of claim
 1. 9. The nucleic acidencoding the FVIII variant as claimed in claim 1, wherein R at saidPACE/furin cleavage site is substituted with an amino acid selected fromthe group consisting of a seine, lysine, methionine, cysteine, prolineand tyrosine.
 10. The nucleic acid encoding the FVIII variant as claimedin claim 1, wherein at least one amino acid at said PACE/furin cleavagesite is deleted.
 11. The nucleic acid encoding the FVIII variant ofclaim 1, wherein two of the amino acids at the PACE/Furin cleavage siteare deleted.
 12. The nucleic acid encoding the FVIII variant of claim 1,wherein three of the amino acids at the PACE/Furin cleavage site aredeleted.
 13. The nucleic acid encoding the FVIII variant of claim 1,wherein all four of the amino acids at the PACE/Furin cleavage site aredeleted.